This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Francisella tularensis is a highly infectious Category A bacterial pathogen that causes tularemia, a potentially life-threatening disease in humans. Critical to Francisella's pathogenesis are its ability to replicate within macrophages and to subvert the host immune response. We recently employed a powerful global in vivo negative selection screen in mice to identify genes required for the pathogenesis of F. novicida, a subspecies of Francisella that causes disease in mice but not humans. This approach resulted in the identification of 164 genes that are required for virulence, 44 of which appear to encode novel virulence factors. We found that 65 of the 164 genes are required for replication in macrophages in vitro. One of the genes with the strongest phenotypes that was essential for replication in macrophages encodes a novel protein of unknown function. A deletion mutant lacking the protein confirmed that this gene (which we refer to as fpeA - Francisella phagosome escape A) is required for F. novicida replication in macrophages as well as virulence in vivo. Electron microscopy revealed that the mutant strain is defective for escape from the macrophage phagosome, explaining its attenuated replication phenotype. This inability to rapidly escape the phagosome and replicate correlated with decreased expression of Francisella pathogenicity island (FPI) genes that are required for these processes. We are currently determining the molecular mechanism by which FpeA controls FPI expression. We are also elucidating the function of several other important virulence genes identified in our screens.